Performance comparison of reversed-phase C18 columns for peptide mapping of monoclonal antibodies

Importance of the Topic

Peptide mapping using reversed-phase C18 liquid chromatography coupled to high-resolution mass spectrometry is essential for the characterization of biotherapeutics. It enables confirmation of primary sequence, detection of post-translational modifications (PTMs) such as deamidation, oxidation, and glycosylation, and monitoring of critical quality attributes (CQAs) that impact safety and efficacy.

Objectives and Overview of Study

This work compared the chromatographic performance of the Thermo Scientific Hypersil GOLD Peptide C18 column against four competitor reversed-phase columns for peptide mapping of monoclonal antibodies. Key goals included evaluating sequence coverage, PTM separation (particularly deamidation in the PENNYK motif), retention of hydrophilic and hydrophobic peptides, carry-over, and lot-to-lot consistency.

Methodology

NISTmAb and rituximab samples were reduced, alkylated, buffer-exchanged, and digested with trypsin. Peptide standards and mAb digests underwent UHPLC separation on 2.1×150 mm C18 columns (particle sizes 1.6–2.7 µm, pore sizes 100–175 Å) under a 70 min gradient at 0.25 mL/min, 50 °C. Data were acquired on an Orbitrap Exploris 240 in positive polarity, full MS at 120 000 resolution and data-dependent MS2. Peptide identification and quantitation used BioPharma Finder and Chromeleon software with stringent filters for mass accuracy, isotopic fit, and confidence thresholds.

Instrumentation

• Thermo Scientific Vanquish Horizon UHPLC system
• Thermo Scientific Orbitrap Exploris 240 mass spectrometer

Main Results and Discussion

Sequence coverage exceeded 97% for heavy and light chains on all columns; Hypersil GOLD Peptide delivered >98% coverage.
The Hypersil column showed superior peak symmetry (A≈1.0–1.3), retention time precision (%CV < 0.6%), and peak area reproducibility (%CV < 5%) compared to competitors.
Chromatographic resolution of the PENNYK Fc peptide and its two deamidated forms achieved R > 1.4 for the first deamidation and R > 2.0 for the second on Hypersil and select vendors, while some competitor columns failed to resolve these species.
Hydrophilic peptides, including VSNK and its deamidated form, eluted with improved retention and distinct peaks on Hypersil GOLD Peptide, whereas other columns showed co-elution near void volume.
Carry-over of hydrophobic tryptic peptides was <1% across all columns; Hypersil exhibited consistent low carry-over (<0.3%).
Targeted quantitation of PTMs (deamidation, succinimide, oxidation, pyroglutamate, C-terminal lysine) demonstrated high repeatability (%CV < 10%) and lot-to-lot consistency for Hypersil GOLD Peptide.

Benefits and Practical Applications

• Robust separation of diverse peptide chemistries supports comprehensive quality assessment.
• High resolution of critical PTM hotspots ensures confident detection and quantitation.
• Excellent lot-to-lot reproducibility facilitates implementation in regulated QC environments.
• Low carry-over and stable retention times enhance throughput and reliability.

Future Trends and Applications

Emerging workflows will integrate multi-attribute methods (MAM) to simultaneously monitor multiple CQAs. Continued development of column chemistries may further improve retention of extremely hydrophilic peptides and enable rapid glycopeptide profiling. Advances in automation, high-throughput UHPLC-MS platforms, and artificial intelligence-driven data analysis will drive deeper insight into biotherapeutic heterogeneity and accelerate process development.

Conclusion

The Hypersil GOLD Peptide reversed-phase column delivers outstanding chromatographic performance for monoclonal antibody peptide mapping, outperforming or matching competitor columns in sequence coverage, PTM resolution, hydrophilic peptide retention, reproducibility, and lot-to-lot consistency. It is well suited for advanced biopharma workflows and QC applications.

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